Expanding genotype/phenotype of neuromuscular diseases by comprehensive target capture/NGS Xia Tian, PhD* ABSTRACT * Wen-Chen Liang, MD Objective: To establish and evaluate the effectiveness of a comprehensive next-generation * Yanming Feng, PhD sequencing (NGS) approach to simultaneously analyze all genes known to be responsible for Jing Wang, MD the most clinically and genetically heterogeneous neuromuscular diseases (NMDs) involving spi- Victor Wei Zhang, PhD nal motoneurons, neuromuscular junctions, nerves, and muscles. Chih-Hung Chou, MS Methods: All coding exons and at least 20 bp of flanking intronic sequences of 236 genes causing Hsien-Da Huang, PhD NMDs were enriched by using SeqCap EZ solution-based capture and enrichment method fol- Ching Wan Lam, PhD lowed by massively parallel sequencing on Illumina HiSeq2000. Ya-Yun Hsu, PhD ; 3 Thy-Sheng Lin, MD Results: The target gene capture/deep sequencing provides an average coverage of 1,000 per Wan-Tzu Chen, MS nucleotide. Thirty-five unrelated NMD families (38 patients) with clinical and/or muscle pathologic Lee-Jun Wong, PhD diagnoses but without identified causative genetic defects were analyzed. Deleterious mutations Yuh-Jyh Jong, MD were found in 29 families (83%). Definitive causative mutations were identified in 21 families (60%) and likely diagnoses were established in 8 families (23%). Six families were left without diagnosis due to uncertainty in phenotype/genotype correlation and/or unidentified causative Correspondence to genes. Using this comprehensive panel, we not only identified mutations in expected genes but Dr. Wong: also expanded phenotype/genotype among different subcategories of NMDs. [email protected] or Dr. Jong: Conclusions: Target gene capture/deep sequencing approach can greatly improve the genetic [email protected] diagnosis of NMDs. This study demonstrated the power of NGS in confirming and expanding clin- ical phenotypes/genotypes of the extremely heterogeneous NMDs. Confirmed molecular diagno- ses of NMDs can assist in genetic counseling and carrier detection as well as guide therapeutic options for treatable disorders. Neurol Genet 2015;1:e14; doi: 10.1212/NXG.0000000000000015 GLOSSARY AD 5 autosomal dominant; AR 5 autosomal recessive; BCM 5 Baylor College of Medicine; CACTD 5 carnitine acylcarnitine translocase deficiency; CCD 5 central core disease; CM 5 congenital myopathy; CMD 5 congenital muscular dystrophy; CMT 5 Charcot-Marie-Tooth disease; CMyS 5 congenital myasthenic syndrome; CNM 5 centronuclear myopathy; EDMD 5 Emery-Dreifuss muscular dystrophy; HSAN 5 hereditary sensory and autonomic neuropathy; LGMD 5 limb-girdle muscular dystrophy; MH 5 malignant hyperthermia; MM 5 metabolic myopathy; MTS 5 myotonic syndrome; NGS 5 next-generation sequencing; NM 5 nemaline myopathy; NMD 5 neuromuscular disease; PFIS 5 paralytic floppy infant syndrome; SMARD1 5 spinal muscular atrophy with respiratory distress type 1; VUS 5 variants of unknown significance. Neuromuscular diseases (NMDs) are genetically and clinically heterogeneous. To date, more than 360 genes have been reported to cause NMDs.1 As a group, the combined NMD prev- alence is greater than 1 in 3,000.2 3,4 See editorial The majority of NMDs are inherited, degenerative, and rare. An early definitive molecular diagnosis is crucial for genetic counseling, family planning, prognosis, therapeutic strategies, and Supplemental data at Neurology.org/ng *These authors contributed equally to the manuscript. From the Baylor Miraca Genetics Laboratories (X.T., Y.F., J.W., V.W.Z., L.-J.W.), Houston, TX; Department of Pediatrics (W.-C.L., Y.-J.J.), Department of Laboratory Medicine (Y.-J.J.), and Department of Pathology (W.-T.C.), Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; Graduate Institute of Medicine (Y.-J.J.), College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Molecular and Human Genetics (J.W., V.W.Z., L.-J.W.), Baylor College of Medicine, Houston, TX; Institute of Bioinformatics and Systems Biology (C.-H.C., H.-D.H.), and Department of Biological Science and Technology (H.-D.H., Y.-J.J.), National Chiao Tung University, Hsinchu, Taiwan; Department of Pathology (C.W.L.), The University of Hong Kong, Pokfulam, Hong Kong; and Department of Neurology (T.-S.L.), National Cheng Kung University Medical College and Hospital, Tainan, Taiwan. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge was paid by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially. Neurology.org/ng © 2015 American Academy of Neurology 1 ª 2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. long-term care plans.3–7 The recent develop- NMDs.24 We categorized NMDs and their causative genes into ment of next-generation sequencing (NGS) 10 groups, including MM, CMD, CM, other myopathies, motor neuron disease, CMyS, arthrogryposis multiplex congenita, MTS has accelerated the discovery of novel NMD and ion channel muscle diseases, CMT, and other muscular dystro- – phenotypes and genotypes,8 11 including the phies as listed in table 1. Mitochondrial genes were not included. identification of mutations in 5 large NMD A custom NimbleGen in-solution DNA capture library was genes (TTN, NEB, SYNE1, RYR1, and designed to capture all 4,815 coding exons and at least 20 bp flanking intron regions of the 236 NMD-related genes. The 1,4,8–12 DMD ) (table e-1 at Neurology.org/ng). NM accession numbers of the genes are listed in table e-1. The With the ever-increasing number of causative coding regions were enriched according to manufacturer’s in- genes and clinical heterogeneity, a comprehen- structions (Roche NimbleGen Inc., Madison, WI) and sequenc- ing was performed on HiSeq2000, as previously described.21,25 sive molecular approach with the feasibility to add newly discovered genes for analysis Sequence alignment and analytical pipeline for variant calling. Conversion of raw sequencing data, demultiplexing, in a cost- and time-effective manner is sequence alignment, data filtering, and analyses using CASAVA 1,4,7,12–16 needed. v1.7, NextGENe software were performed as previously A recent study using PCR enrichment and described.21,25 Multiple in silico analytical tools, such as NGS approach to analyze 12 genes known SpliceSiteFinder-like, MaxEntScan, NNSPLICE, and GeneSplicer, were used to predict the effects of splice site variants (Alamut, http:// to cause congenital muscular dystrophy www.interactive-biosoftware.com). SIFT26 and PolyPhen-227 were (CMD) on 26 samples with known muta- used to predict the pathogenicity of novel missense variants. The tions17 reported that 49 exons (15%) had pathogenicity of the variants was categorized according to published , 3 17 databases, such as Human Gene Mutation Database (http://www. insufficient coverage ( 20 ). Among 15 biobase-international.com/product/hgmd), PubMed (http://www. known variants, 6 (40%) were not detected. ncbi.nlm.nih.gov/pubmed), and American College of Medical Similar studies on congenital myasthenic Genetics guidelines.28 The analytical flowchart is depicted in syndrome (CMyS),18 Charcot-Marie-Tooth figure e-1. All mutations and novel variants identified by NGS 21,25 19 were confirmed independently by Sanger sequencing. Family disease (CMT), Duchenne/Becker muscular members, if available, were also tested to evaluate the mode of 20 21,22 dystrophy, and metabolic myopathy (MM) inheritance, disease segregation, and clinical correlation. have demonstrated the clinical utility of NGS in Detection of deletions using sequence read coverage data specific disease categories. Nevertheless, these are from NGS. We used a newly developed analytical method to small-scale studies focusing on subcategories of detect exonic deletions using the same set of NGS data by com- NMDs. Here, we describe a comprehensive paring the normalized coverage depth of each individual exon of the test sample with the mean coverage depth of the same exon target gene capture/NGS approach, analyzing from a group of 20 reference samples.29 236 genes. RESULTS Characteristics of target gene capture and METHODS Standard protocol approvals, registrations, sequence depth. More than 99.4% (4,787/4,815) of and patient consents. This study was conducted according to the target sequences were enriched in an unbiased the Institutional Review Board–approved protocols of both fashion, with a minimal coverage of 203 and a mean Kaohsiung Medical University Hospital, Taiwan, and Baylor coverage depth of 1,1363 per base (figure 1). An College of Medicine (BCM), Houston, TX. A signed informed average of 28 exons per sample was consistently insuf- consent was obtained for each participant. ficiently sequenced (,203) due to the high GC Patients and DNA samples. Patients were clinically evaluated content, sequence homologies in the genome, short in Taiwan, and DNA samples were analyzed at BCM. DNA sam- tandem repeats, or secondary structural difficulties ples from 35 unrelated families (38 patients) with clinical diagnoses of NMD who underwent electrophysiologic examination and (table e-2). muscle imaging and/or muscle biopsies were analyzed. Patients Clinical history. A total of 35 unrelated affected fami- with a proven common genetic diagnosis of spinal muscular atro- lies (38 patients) with clinical diagnosis of NMD were phy, Duchenne muscular dystrophy, myotonic dystrophy types 1 and 2, CMT type 1A, or facioscapulohumeral muscular dystro- studied. Among them, 3 families had 2 affected fam- phy were not included in the study. The initial diagnoses included ily members (patients 4/6, 19/20, and 25/26). The congenital myopathy (CM) (23 patients), CMD (5), limb-girdle male to female patient ratio was about 1 to 1. The muscular dystrophy (LGMD)23 (4), CMT (3), MM (2), and majority of patients (27/38, 71%) presented with par- myotonic syndrome (MTS) and ion channel muscle disease (1).